Biochemical Engineering Journal 67 (2012) 225–230 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal journa l h omepage: www.elsevier.com/locate/bej Regular article A label-free immunosensor based on recordable compact disk chip for early diagnostic of the dengue virus infection Igor T. Cavalcanti a , Maria I.F. Guedes b , Maria D.P.T. Sotomayor c , Hideko Yamanaka c , Rosa F. Dutra a,∗ a Laboratório de Engenharia Biomédica, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-420, Recife, PE, Brazil b Laboratório de Bioquímica Humana, Universidade Estadual do Ceará, Fortaleza, CE, Brazil c Instituto de Química de Araraquara, Universidade Estadual Paulista Julio de Mesquita Filho, Araraquara, SP, Brazil a r t i c l e i n f o Article history: Received 1 December 2011 Received in revised form 17 June 2012 Accepted 23 June 2012 Available online 3 July 2012 Keywords: Electrochemical immunosensor NS1 Dengue CD-trode a b s t r a c t The non-structural protein 1 (NS1) of dengue virus is abundantly circulating in the blood during the acute phase of the dengue infection being correlated with viremia levels and hence can be used to early diagnostic of the dengue hemorrhagic fever. An electrochemical immunosensor based on gold film elec- trode obtained from a recordable compact disk (CD-trode) was developed for NS1 protein. Anti-NS1 monoclonal antibodies were immobilized on the CD-trode via protein A. The stepwise immobilization of the anti-NS1 was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The analytical response to the NS1 interaction with anti-NS1 immobilized on CD-trode was detected by applying the differential pulse voltammetry technique. The immunosensor showed a linear response from 1 to 100 ng/mL of NS1 and a detection limit of 0.33 ng/mL. This label-free immunosensor exhibited a good reliability for NS1 detection in serum samples presuming an early diagnostic of the dengue virus. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Dengue virus infection remains an important public health problem worldwide, particularly in tropical and sub-tropical areas [1]. The severe forms of dengue virus infection include dengue hemorrhagic fever and dengue shock syndrome, which can lead to profound shock and death [2]. The diagnosis of dengue infection remains an open question because current methods have limi- tations, such as high cost and low detection accuracy [3]. The non-structural protein 1 (NS1) of dengue virus circulates in human serum mostly from days 1–6 after the onset of clinical symptoms, with the peak NS1 antigen detection occurring between days 3 and 5 in both primary and secondary infections [4]. NS1 is abundantly circulating in blood during the acute phase of the disease and hence can also be used to diagnose patients at the risk for developing dengue hemorrhagic fever being a benefit for early rapid diagnosis of the dengue infection [5]. Thus, developments of rapid and simple analytical methods for NS1 detection are desirable. Compared with conventional immunoassay methods, the biosensors based on electrochemical transducers have gained great attention for clinical diagnostics, since they present simple instrumentation, high sensitivity, low cost and are also easily com- patible with techniques of microfabrication [6]. Electrochemical immunosensors which do not require a label or tracer conjugated ∗ Corresponding author. Tel.: +55 81 2126 8200; fax: +55 81 2126 8000. E-mail addresses: rosa.dutra@ufpe.br, rosa.dutra@pq.cnpq.br (R.F. Dutra). to antibodies or antigens for electrochemical detection are more attractive [7]. Label-free immunosensors with higher specificity and accuracy are achieved when high-affinity antibodies are used and, in particular, when an appropriated immobilization method to control the orientation of biomolecules is employed [8]. An ideal immobilization method ensures improved specificity, espe- cially when dealing with a more complex sample matrix such as urine, cerebral spinal fluid (CSF), and serum, which contains high levels of serum albumin and immunoglobulins [9]. Protein A is well- known due to its binding specificity for Fc region of heavy chains of antibodies. Since antibodies are immobilized in oriented way by exposing their Fab active sites to epitopes, the sensor selectivity are strongly improved. One of typical procedures used to production of the gold micro- electrodes consists in sputtering or etching-process on the sensor surface [10]. The quality of the metal layer deposited depends on many factors such as the cleanness of the clean rooms, previous deposit of another material for a better adhesion and an appropri- ate pressure control [11]. However, there are few electrochemical laboratories that possess equipments for these procedures. More- over, metallic films prepared by sputtering on different substrates mainly those produced for construction of disposable electrodes, can be readily derived from recordable compact disks (CD-Rs) [12]. The film sputtering process on the gold films on CD-Rs is very well established in the CD record’s industry, which produces devices with a gold layer varying from 50 to 100 nm [13]. Regarding the simplicity of construction, the gold electrodes prepared from gold CD-Rs (CD-trodes) are very attractive because they present 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.06.016